The present invention relates generally to illumination for microscopy applications, including both fluorescence microscopy and general microscopy applications, and specifically to an illumination apparatus that uses phosphor emissions to provide broad-spectrum white light. By using multiple phosphor types, the illumination apparatus provides a broad-spectrum light output that is highly suitable for exciting the large variety of fluorescent dyes that are used in fluorescence microscopy applications, from a single illuminator. In addition, the illuminating apparatus can provide high-quality white light for brightfield viewing in general microscopy applications, including visible light image capture and photography.
Fluorescence microscopy is popularly used in numerous bio/medical applications since it enables users to label and observe specific structures or molecules. Briefly, fluorescence is a chemical process in which light of a specific wavelength or wavelength range is shined upon a fluorescent molecule, causing electrons from said fluorescent molecule to be excited to a high energy state, in a process known as excitation. These electrons remain briefly in this high energy state, for roughly a nanosecond, before dropping back to a low energy state and emitting light of a longer wavelength. This process is referred to as fluorescent emission, or alternatively as fluorescence.
In a typical fluorescence microscopy application, one or more types of fluorescent materials or molecules (also referred to as fluorescent dyes) are used, along with an illuminator apparatus that provides the exciting wavelength, or wavelengths. Different fluorescent molecules or dyes can be selected to have visually different emission spectra. Since the different fluorescent molecules or dyes that are typically used in fluorescence microscopy applications typically have different excitation wavelengths, they can be selectively excited so long as the bandwidth of the excitation light for one fluorescent molecule or dye does not overlap the excitation wavelengths of other fluorescent molecules or dyes that are being used in the same experiment. This is typically achieved by using specific wavelength-range bandpass filters to create narrow bandwidth excitation light. Broadband excitation light may also be used to simultaneously excite multiple fluorescent dyes. Furthermore, fluorescence is a probabilistic event with low signal levels so an intense light is typically used to increase the chances of the process occurring. Most fluorescence microscopy applications also benefit from having a uniformly intense illuminated field of view or area, ideally such that the size and shape of the illuminated area can be modified. Simultaneously achieving all these criteria has been difficult, but is necessary for current and future applications that require increasing levels of illumination control and consistency.
Traditional prior art fluorescence microscopy illuminators have relied on metal halide arc lamp bulbs such as Xenon or Mercury bulbs, as light sources. The broad wavelength spectrum produced by these lamps, when combined with specific color or bandpass filters, allows for the selection of different illumination or excitation wavelengths. Alternatively, multiple fluorescent dyes, with different excitation and emission wavelengths, may be simultaneously excited. In this type of implementation using metal halide arc lamp bulbs, the speed with which different wavelengths can be selected is limited by the mechanical motion of moving various filters into place. In addition to the sluggishness and unreliability of filter wheels, metal halide arc lamps are also hampered by the limited lifetime of the bulb, typically ˜2000 hours. The intensity of the light output declines with bulb use and once exhausted, the user has to undergo a complicated and expensive process of replacing the bulb and subsequently realigning the optics without any guarantee that the illuminator will perform as before. These disadvantages make acquiring consistent results difficult and inconvenient for users who must deal with the variable output of the bulbs, and who must either be trained in optical alignment or call upon professionals when a bulb needs to be replaced. In addition, metal halide arc lamps produce substantial heat, including radiated emissions in the infrared region that can cause heating of the illuminated specimens. This can lead to specimen damage, especially in the case of biological specimens. Similarly, radiated emissions in the UV region may also harm specimens. (In both cases, the use of appropriately designed excitation filters can prevent specimen exposure to damaging wavelengths.)
In recent years, several prior art multiple wavelength illuminators have been developed using different colored LEDs as light sources, that overcome numerous limitations of metal halide arc lamps. Not only do they last longer, with the lifetime of an LED chip being typically rated at well over 10,000 hours, but in addition the power output varies negligibly over that period. Furthermore, the bandwidth of the spectral output of an LED chip is typically narrow (<30 nm) which may eliminate the need for additional bandpass filters. The intensity of the output light can be quickly and accurately controlled electronically by varying the current through the LED chip(s), whereas in metal halide illuminators, the output intensity of the bulb is essentially fixed, and apertures or neutral density filters are used to attenuate the light entering the microscopy.
Prior art LED illuminators for fluorescence microscopy have thus far used up to 5 separate LED modules, each containing one, up to a few chips, for each wavelength. Since the LED chips in these modules have their own individual packaging, the modules are large so that light beams emitted from the modules will need to be combined using optical elements. Although such prior art LED illuminators allow the user the flexibility to swap out modules for new modules with different wavelengths, the additional elements such as lenses, mirrors and heat sinks required for each separate color add complexity, bulk and cost. Furthermore, the long optical paths required to combine the beams from multiple LED chips or modules that are spatially separated, make it difficult to collect and shape already highly divergent light coming from the LED chips. Even when multiple LEDs are packaged or mounted close to each other, the light output of LED chips that are located even a short distance away from the optical axis will be poorly coupled to the objective lens of the microscope.
Another limitation of prior art LED illuminators for fluorescent microscopy is that there is a “dead zone” in the visible light spectrum, where LED chips are either not readily available, or are of very limited optical output. This dead zone is roughly in the portion of the visible light spectrum that lies between green and amber (or orange), in the approximate wavelength range of 540-595 nm. Unfortunately, several popular fluorescent dyes require excitation light that is in this dead zone.
These practical issues have limited the application of such illuminators in fluorescence microscopy, which in general requires light that is both intense and spatially uniform, across the full range of wavelengths that are required for the excitation of popular fluorescent dyes.
Although the narrow spectral bandwidth (typically <30 nm) of individual LEDs can be an advantage in some fluorescent microscopy applications, bandpass excitation filters may still be needed, in order to more closely match the excitation wavelength requirements of the dye(s) being used. If excitation filter(s) are being used anyway, then the narrow spectral bandwidth of LEDs can become a disadvantage, in that the LED wavelengths being used must be selected to match the types of dyes being used. For this reason, it is desirable to have a broad-spectrum illumination apparatus that provides the lifetime, reliability, and other advantages of an LED illumination apparatus.
In order for LED illuminators and light engines to act as a satisfactory replacement for illuminators used in general microscopy applications, such as brightfield illuminators, it is desirable and even necessary to produce white light with characteristics that are similar to the light produced from an incandescent bulb, or in some cases, to accurately replicate the light provided by natural sunlight. This is especially important for microscopy applications that demand high quality light with well-controlled parameters. This is true for human eye viewing, as well as microscope photography and imaging. In a general sense, this means that the LED illuminator or light engine should have a broad spectral response or characteristic that mimics the spectral response of an incandescent bulb, and/or natural sunlight.